As studies relating to protein molecular structures have become increasingly important in all biomedical fields, it has become necessary to grow improved, highly ordered, single protein crystals which will exhibit higher resolution during protein crystallography. High quality crystals allow researchers to define protein molecular structures in greater detail. Knowledge gained through protein crystallography with X-ray diffraction and computer modeling is invaluable for applications in areas of drug design, protein engineering, material science, agriculture, as well as for the establishment of structural and functional foundations of biochemistry and molecular biology.
To date, the art of growing protein crystals has been a relatively inexact science because of the difficulties in controlling the many parameters which affect protein crystal growth, such as protein and precipitant concentrations, temperature, degree of alkalinity and acidity, and equilibration rate. Computer technology, on the other hand, has reduced the time necessary to interpret the results of protein crystallography on a particular crystal from a period of months to, in most cases, a few days. As a result, more crystals can be studied in a given time frame, which directs more emphasis toward the ability to grow improved, more uniform protein crystals as a matter of routine.
One method which is used to grow protein crystals is the liquid diffusion method in which a protein solution and a precipitant solution are made to come into contact with each other and are allowed to mix by diffusion to produce the condition in which the protein becomes less soluble and begins to crystallize. Since liquid-liquid diffusion in a gravity environment is disrupted by convection currents, this method has achieved only limited success.
Another method used to grow protein crystals is the dialysis method in which a solution containing dissolved protein to be crystallized is contained behind a dialysis membrane which separates it from a precipitant solution. This semi-permeable membrane allows small solute molecules and ions to pass through, while larger protein molecules remain contained. The protein container having the dialysis cover is immersed in the precipitant solution, which has a relatively high concentration of solute, such as sodium chloride or methyl pentane diol, resulting in a concentration gradient between the precipitant solution and the protein solution. This gradient causes solute from the precipitant solution to move through the membrane into the protein solution. As solute concentration increases in the protein solution, the dissolved protein becomes less soluble and crystal nucleation occurs, forming nuclei of small crystallites which increase in size to produce the desired crystals. In this method, however, there are typically no provisions to control the rate at which solute moves through the membrane, meaning that the rate of crystal growth cannot be precisely controlled.
One of the primary methods of growing protein crystals is the hanging drop method in which a droplet of protein and precipitant (such as sodium chloride or methyl pentane diol) solution is suspended over a reservoir containing a precipitant solution of relatively high concentration within a gas-tight container. The difference in vapor pressure between the droplet and the reservoir causes water vapor to be transported through the vapor phase from the droplet to the reservoir, thus decreasing the droplet size as the droplet attempts to reach vapor pressure equilibrium. In turn, this concentrates the dissolved protein, and as the protein becomes less soluble, crystal nucleation occurs, forming crystallites which increase in size to produce the desired crystals. Problems with this method are a lack of control of the rate at which a droplet size is changed and the inability to increase drop size.
It is, therefore, an object of this invention to provide an apparatus which will allow an operator or control system to control the rate at which drop size is modified and to modify drop size by either moving solvent (usually water) from the drop or by adding solvent to the drop via the vapor phase.